Antisense oligonucleotides have demonstrated potential as new types of therapeutic agents for treating such diseases and disorders as viral diseases, cancer, genetic disorders, as well as other diseases and disorders.sup.1. Extensive research has been carried out and is being continued in industrial and academic laboratories to explore this potential.sup.2.
A problem that has been encountered with the approach of utilizing antisense oligonucleotides as therapeutic agents is related to the selectivity of the agents in vivo. In view of the low concentrations of intracellular polynucleotide targets and the low concentrations of therapeutic oligonucleotides that can be introduced into cells, it is recognized that there is a need for oligonucleotides with high binding affinities. The binding affinity is related to the length of the oligonucleotides, preferably 20-mers and longer. But, in the case of long oligonucleotides, a mismatch in base pairing is less destabilizing then in the case of a short oligonucleotide. Hence, the desired destabilizing effect is lessened by the use of longer oligonucleotides while the selectivity is increased.
Experts have noted that "high sequence specificity" and "high affinity" are contradictory demands.sup.3. It has further been concluded that on the basis of the extent to which antisense oligonucleotides can cause cleavage of RNAs at imperfectly matched target sites, in systems that were tested it was probably not possible to obtain specific cleavage of an intended target RNA without also causing at least the partial destruction of many non-targeted RNAs.sup.4. Hence, experts in the field, based on conducted research, have concluded that the conflicting requirements of specificity and affinity are major hurdles to overcome. Several methods have been reported for covalently linking oligonucleotide blocks in aqueous media.sup.5a-l. All of these methods require an additional chemical agent to yield a stable ligated product. Depending on the approach, the added reagent may be an "activating agent" such as a water soluble carbodiimide or cyanoimidazole.sup.5a-k or it may be a reducing agent such as sodium cyanoborohydride.sup.51. In either case, the need for the third reagent precludes chemical ligation in vivo since such compounds are toxic, react with water, and could not be introduced into living systems in sufficient amounts to bring about the desired coupling reaction.
The present invention provides a novel method for covalently linking oligonucleotide blocks present in low concentrations in an aqueous medium without need for an additional condensing or stabilizing reagent. It therefore opens the door for in situ chemical ligation in living systems. Since the reactions are greatly accelerated in the presence of a complementary oligonucleotide sequence, one should in principle be able to form long oligonucleotide strands selectively in vivo when a target polynucleotide (e.g. m-RNA or DNA from a virus or cancer cell) containing consecutive nucleotide sequences complementary to the individual oligonucleotide strands is present. Long oligonucleotide strands, which bind with high affinity, would therefore be generated in situ from shorter strands that bind with lower affinity, when the target polynucleotide is present. This invention could therefore solve the problem of the conflict of achieving high affinity as well as high specificity, in therapeutic and also in diagnostic applications.